Background
Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects, which contribute to neuronal damage and inflammation [
1,
2]. It has been well-recognized that high doses of METH impair nigrostriatal dopaminergic systems in both rodents [
3] and primates [
4]. Although the pathogenesis of METH-induced dopaminergic neurotoxicity remains to be elucidated, this neurotoxicity may be, at least in part, related to oxidative stress [
5,
6], inflammatory changes [
7], and apoptosis [
8]. METH may act upon neurons as a central processor of inflammation by releasing pro-inflammatory molecules [
9]. These pro-inflammatory molecules may further activate downstream apoptotic signaling pathways in neurons, ultimately resulting in neuronal death and/or the activation of glial cells, which can further exacerbate neuroinflammation. In addition, the pathogenesis of dopaminergic neurotoxicity observed in Parkinson’s disease (PD) is similar to the neuroinflammatory and neurotoxic effect of METH [
10].
Several studies have reported the neurotoxic mechanism of METH, including oxidative stress and apoptosis in neuronal cells [
11‐
13]. METH causes induction of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α [
1,
14,
15]. Specifically, TNFα has been reported to be a potent stimulator of IL-6 production, whose pleiotropic action can be through the TNF receptor (TNFR) [
9]. The activation of TNFR stimulates several signaling pathways that regulate cellular processes, ranging from cell proliferation and differentiation to cell death [
16]. With respect to IL-6, its production seems to be regulated by several signaling cascades [
9,
17], including TNF-α activation via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT) signaling pathways [
14,
15,
18].
Increasing evidence has revealed that METH-induced neurodegeneration is associated with mitochondria-dependent apoptosis [
19]. In addition, administration of METH in mice has been shown to cause an increase in pro-apoptotic proteins (BAX, BAD, and BID) but a decrease in anti-apoptotic Bcl-2-related proteins [
11]. METH-induced alterations in these proteins have been suggested to form channels that result in mitochondrial membrane potential loss and allow cytochrome c release [
20]. These observations propose that mitochondrial damage may contribute to METH-induced neurotoxicity. A direct cytotoxic role of METH has been found to be mediated by the mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspases and the induction of apoptosis [
21]. Although extensive research has focused on development of pharmacological agents that inhibit METH-induced neurotoxicity, FDA-approved pharmacotherapies for the treatment of negative effects of METH are still lacking [
22]. Novel approaches aimed at overcoming the negative effects of METH are urgently needed in this field.
Asiatic acid (AA), a natural pentacyclic triterpene derived from the medicinal herb
Centella asiatica, elicits multiple bioactivities, including antioxidant, anti-apoptotic, and anti-inflammatory properties [
23‐
26]. A previous study showed that AA attenuates glutamate-induced cognitive deficits in mice and apoptosis in human neuroblastoma dopaminergic SH-SY5Y cells [
27]. AA improves learning and memory in an animal model, which was correlated with an increase in hippocampal neurogenesis [
28‐
30]. Although studies on the physiological function of AA, including anti-inflammatory and anti-apoptotic functions, were performed, the effectiveness of AA related to METH-induced neurotoxicity has not been evaluated. Therefore, in this study, we investigated the potential therapeutic effects of AA on the production of pro-inflammatory cytokines induced by METH. We also evaluated the molecular impact of AA on the signal transduction pathways involved in METH-induced neurotoxicity.
Methods
Cell cultures and reagents
A dopaminergic human neuroblastoma cell line SH-SY5Y (America Tissue Culture Collection, CRL-2266; ATCC, VA, USA) was cultured in a Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% Anti-Anti (Gibco). BV-2 murine microglia cells, a generous gift from Dr. Hoe (Korea Brain Research Institute, KBRI), were cultured in DMEM containing 10% FBS and 0.1% gentamicin (Gibco). Mesencephalic neuron cultures were prepared from the ventral mesencephalic tissues of embryonic day 13–14 rats, as described previously [
31]. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Keimyung University (Daegu, South Korea; EXP-IRB number: KM_2016-007; the date of approval: 1 April 2016) in accordance with the criteria outlined in the Institutional Guidelines for Animal Research. Briefly, dissociated cells were seeded at 1 × 10
5/well to poly-
d-lysine- and laminin-coated 24-well plates. Cells were cultured in a Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (Gibco) containing ITS premix (Sigma-Aldrich, MO, USA) and 1% penicillin-streptomycin (Gibco). Cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO
2.
Methamphetamine (METH) was purchased from the Ministry of Food and Drug Safety (Korea). Asiatic acid (AA) was purified and received from Dr. Ki Yong Lee, a professor of the College of Pharmacy, Korea University. Signal inhibitors were obtained from Sigma-Aldrich (MO, USA).
Morphological examination
Morphological changes in cells were observed under an inverted phase-contrast microscope (Leica, Germany). The effect of AA on METH-induced neuroinflammation was observed for 24 h, and METH-induced neurotoxicity was observed for 12 h. The photographs were taken at ×200 or ×400 magnification using a digital camera.
Cytotoxicity assay
Cells were plated in 96-well culture plates at 1 × 106 cells/ml in culture medium and allowed to attach for 24 h. Media were then discarded and replaced with new medium containing various concentrations of METH and AA. The cells were cultured for an additional 24 h, and then, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT, 5 mg/ml; Sigma-Aldrich) was added to each well (1/10 of medium volume), and the samples were incubated at 37 °C in a 5% CO2 incubator for 4 h. The formazan precipitate was dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 540 nm using a microplate reader (Bio-Rad Laboratories, CA, USA).
Treatment kinase inhibitors and siRNA transfection
Cells were pretreated with various inhibitors (Sigma-Aldrich) such as NF-κB-specific inhibitor: Bay11-7085 (20 μM), STAT3-specific inhibitor: S3I-201 (20 μM), and ERK1/2-specific inhibitor: PD98059 (20 μM). After 1 h, the cells were treated and co-cultured with METH for 24 h. Cells were transfected with control siRNA (Cat no: sc-37007, Santa Cruz, CA, USA) and ERK-MAPK siRNA (Cat no: 6560, Cell Signaling Technology, MA, USA) using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instruction.
Enzyme-linked immunosorbent assay (ELISA)
The culture medium of the cells was harvested, and cytokine production (TNFα and IL-6) in the supernatant was measured with a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) using a Quantikine TNF-α and IL-6 kit (R&D systems, MN, USA) according to the manufacturer’s instructions.
Immunoblot analysis
Cytosolic and nuclei protein fractions were obtained as described [
32]. The protein concentration was determined with a Bio-Rad Bradford kit (Bio-Rad Laboratories, CA, USA). The samples were boiled for 5 min, and equal volumes were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred onto a nitrocellulose membrane and probed with anti-TNFR, anti-p-NF-κB p65, anti-NF-κB p65, anti-p-STAT3, anti-STAT3, anti-p-JAK2, anti-JAK2, anti-p-ERK1/2, anti-ERK1/2, anti-p-JNK1/2, anti-JNK1/2, anti-p-p38, anti-p38, anti-TH, anti-lamin B, and anti-β-actin (all from Cell Signaling Technology) followed by a secondary antibody conjugated to horseradish peroxidase and detected with enhanced chemiluminescence reagents (Amersham Bioscience, UK). The luminescent signals were analyzed using an ImageQuant LAS 4000 Scanner of GE Healthcare (Piscataway, NJ, USA).
Reverse-transcription and real-time PCR
Total RNA was extracted from neuronal cells with TRIzol reagent (Invitrogen Co, Grand Island, NT, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized with oligo-d(T) primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time PCR was performed in a CFX 96 Touch™ detection system (Bio-Rad Laboratories) using SYBR Green PCR Master Mix (Bio-Rad Laboratories). Each measurement was repeated at least in triplicate, and the relative quantity of target mRNA was determined using the comparative threshold (Ct) method by normalizing target mRNA Ct values to those for β-actin (∆Ct). Aliquots of cDNA were used for PCR using primer sets specific to TNF-α, IL-6, and β-actin as a control. Used primers are as follows: for TNF-α, 5′-TCT CGA ACC CCG AGT GAC AA-3′ (sense) and 5′-TGA AGA GGA CCT GGG AGT AG-3′ (antisense); for IL-6, 5′-CAC AGA CAG CCA CTC ACC TC-3′ (sense) and 5′-TTT TCT GCC AGT GCC TCT TT-3′ (antisense); and for β-actin, 5′-CTT CCT GGG CAT GGA GTC CT-3′ (sense) and 5′-GGA GCA ATG ATC TTG ATC TT-3′ (antisense).
Electrophoretic mobility shift analysis (EMSA) and supershift assay
As described in our previous studies, the DIG Gel Shift Kit (Roche, Mannheim, Germany) was used to detect NF-κB-p65- and STAT3-binding activity, with the instructions of the manufacturer [
32,
33]. The binding activity of NF-κB-p65 and STAT3 in the METH-induced cells was confirmed by EMSA or supershift assay with a DIG-labeled oligonucleotide (NF-κB: 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, STAT3: 5′-CTT CAT TTC CCG TAA ATC CCT AAA GCT-3′) and NF-κB-p65 and STAT3 antibody (Cell Signaling Technology). The NF-κB-p65-oligo, NF-κB-p65-Ab, STAT3-oligo, and STAT3-Ab complexes were separated by electrophoresis on 6% non-denaturing polyacrylamide gels. After electrophoresis, the gels were transferred to nylon membranes and detected by chemiluminescence. The luminescent signals were analyzed on an ImageQuant LAS 4000 Scanner (GE Healthcare).
Immunofluorescence staining
Cells were grown on chamber slides and coating cover slides and fixed with 3.7% paraformaldehyde in PBS. The membrane was permeabilized by treating cells for 2 min with 0.1% Triton X-100 in PBS. The cells were then placed in blocking solution (5% FBS in PBS) at room temperature. Cells were incubated with primary antibodies for 1 h at room temperature. After washing, they were incubated with the Alexa Flour 488 (excitation/emission = 495/519 nm, green, Invitrogen, CA, USA) and Alexa Flour 594 (excitation/emission = 590/617 nm, red, Invitrogen, CA, USA) for 30 min at room temperature. Cells were counterstained with Hoechst 33342 (excitation/emission = 330–380 nm/460 nm, ImmunoChemistry, MN, USA). Slides were mounted using ProLong® Gold antifade reagent (Molecular Probes® by Life Technologies™, CA, USA). Primary antibodies utilized are the following: anti-pNF-κB, anti-pSTAT3, and anti-tyrosine hydroxylase (TH) (from Cell Signaling Technology). Immunolabeling was examined using an Eclipse Ti-U and confocal microscope (Nikon, Tokyo, Japan).
Annexin V/propidium iodide (PI) staining
Apoptotic cells were differentiated from viable or necrotic cells using a combined staining of annexin V-FITC and PI (Invitrogen). To investigate the neurotoxic effect of AA on METH, cells were pretreated with 20 μM AA for 1 h and then exposed to 1.5 mM METH for 12 h. After the cells were trypsinized, they were washed in PBS and resuspended in annexin-binding buffer. The cell suspension (1 × 105 cells/ml) was incubated with FITC-conjugated anti-annexin V and PI for 15 min in the dark. Cells were counted using a BD FACSVerse™ (BD Biosciences, NJ, USA) and analyzed by Flowing 2.5 version software. A total number of 10,000 events were recorded for each sample. The fluorescence spectrum of annexin V and PI were detected using a 527/32-nm and a 586/42-nm filter, respectively.
JC-1 mitochondrial transmembrane potential assay
To measure the mitochondrial transmembrane potential, JC-1 dye (Sigma-Aldrich), a sensitive fluorescent probe, was used. Fluorescence microscopy with a 488-nm filter was used for the excitation of JC-1. Emission filters of 535 and 595 nm were used to quantify the population of mitochondria with green (JC-1 monomers) and red (JC-1 aggregates) fluorescence, respectively. Immunolabeling was examined by an Eclipse Ti-U microscope (Nikon) and BD FACS Verse flow cytometer.
Statistical analysis
All values are expressed as mean ± standard error of the mean (SEM). All data analysis was performed with the GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA) using either a one-way ANOVA with Tukey’s post hoc test for multiple comparisons, with p < 0.05 defined as significant. For the quantification of immunofluorescence, staining results are expressed as percentages of total cells stained with Hoechst 33342 per each field (n = number of fields). For all data, n corresponds to the number of independent experiments.
Discussion
Methamphetamine (METH) can act upon the neurons as a central processor of inflammation by releasing pro-inflammatory molecules [
7]. These pro-inflammatory molecules may further activate downstream apoptotic signaling pathways in neurons, ultimately resulting in neuronal death and/or the activation of glial cells, which can further exacerbate neuroinflammation [
2]. Therefore, inhibition of neurotoxicity and neuroinflammation by METH is important. Pro-inflammatory cytokines TNFα and IL-6 are key mediators of inflammatory responses associated with various neurological disorders including Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis [
14]. A high dose of METH has been shown to induce TNFα and IL-6 in the striatum and hippocampus [
1].
The major findings of this study are as follows: First, asiatic acid (AA) inhibits METH-induced TNFR overexpression and pro-inflammatory cytokine production in the dopaminergic SH-SY5Y cells. Second, METH-induced NF-κB and STAT3 translocation and ERK phosphorylation are prevented by AA pretreatment in dopaminergic SH-SY5Y cells, mesencephalic neurons, and BV2 cells. Third, AA prevents METH-induced neurotoxicity through the mitochondria-dependent apoptotic pathway in dopaminergic SH-SY5Y cells.
TNFα is considered a potent stimulator of IL-6 production, whose pleiotropic action can be triggered through TNFR [
9]. METH significantly increased the TNFR expression in dopaminergic SH-SY5Y cells. However, AA attenuated the elevation of METH-mediated TNFR expression in a concentration dependent.
METH has been found to be associated with neurotoxicity mediated by increased expression of pro-inflammatory cytokines such as TNFα and IL-6 [
14]. These cytokines have also been suggested to play an important role in METH-induced brain dysfunction [
1]. Our result showed that AA inhibits METH-induced secretion of TNFα and IL-6 and their mRNA expression in SH-SY5Y cells, mesencephalic neurons, and BV2 cells.
METH is able to induce pro-inflammatory cytokines and mediators through the NF-κB and STAT3 pathways in dopaminergic and microglia cells [
9,
15,
39]. The potential involvement of NF-κB and STAT3 was investigated because the promoters of both TNFα and IL-6 contain binding sites for NF-κB and STAT3, which are known to be involved in neurological disorders associated with increased inflammation [
14,
18]. In accordance with these findings, our results showed that AA effectively inhibits nuclear translocation of NF-κB (p65) and STAT3 in METH-stimulated dopaminergic SH-SY5Y cells, mesencephalic neurons, and BV2 cells. Thus, AA is likely to be a potent inhibitor of NF-κB and STAT3 signaling.
Besides, MAPK signaling pathways play key roles in the induction of inflammatory cytokines by METH [
40]. METH has been shown to affect phosphor-ERK and p38 MAPK [
41] and monocyte-derived dendritic cells [
42]. ERK is a kinase that plays a role in regulating neuronal and behavioral processes mediated by dopamine and glutamate pathways [
43]. Our results indicated that AA strongly inhibits METH-induced phosphorylation of ERK. However, AA did not participate in the inhibition of phosphorylation of p38.
Several lines of evidence suggest an important role of the intracellular signal transduction pathways in the mechanism of neural plasticity in response to drug abuse [
44]. One of these signal transduction pathways is the ERK1/2 cascade, a member of the mitogen-activated protein kinase (MAPK) family. ERK1/2 activation can phosphorylate tyrosine hydroxylase (TH) and stimulate dopamine synthesis in the brain [
45]. Accordingly, we found that phosphor-ERK was most pronounced among all MAPKs after METH treatment. AA completely suppressed TH and phospho-ERK expression in TH-positive mesencephalic neurons. These results suggest that AA regulated TH expression by the inhibition of ERK by METH.
METH-induced cell death contributes to the pathogenesis of neurotoxicity [
46,
47]. Previous studies have suggested that apoptosis is a critical phenomenon involved in METH-induced neurotoxicity [
11]. Putative mechanisms of METH-induced neurotoxicity have been proposed, including oxidative stress, excitotoxicity, and mitochondrial dysfunction followed by activation of caspase-3 with subsequent cleavage of PARP and DNA fragmentation, appearance of apoptotic cells, and dopaminergic degeneration [
3,
48]. We evaluated the effect of AA on METH-induced neurotoxicity in dopaminergic SH-SY5Y cells to gain a better understanding of the molecular mechanisms involved in AA’s anti-apoptotic effects. AA protected dopaminergic SH-SY5Y cells from apoptosis, as evaluated by assays of cell cycle arrest and mitochondrial membrane potential. Our results demonstrated that METH-induced cleavage of PARP was decreased through the inhibition of caspase-3 cleavage by AA. In addition, Bcl-xL was upregulated, whereas Bax was downregulated by AA in METH-induced neurotoxicity. The protective effects became more pronounced when the dopaminergic SH-SY5Y cells were treated with AA.
Acknowledgements
Asiatic acid was purified and received from Dr. Ki Yong Lee, a professor of the College of Pharmacy, Korea University. BV-2 microglia cells and mesencephalic neuron cultures protocols was provided from Dr. Hoe Hyang-Sook, Korea Brain Research Institute, KBRI.